L-Type Cav 1.2 Calcium Channel-Α-1C Regulates Response to Rituximab in Diffuse Large B-Cell Lymphoma

Published OnlineFirst March 1, 2019; DOI: 10.1158/1078-0432.CCR-18-2146

Translational Cancer Mechanisms and Therapy Clinical Cancer Research L-Type Cav 1.2 Calcium Channel-a-1C Regulates Response to Rituximab in Diffuse Large B-Cell Lymphoma Jiu-Yang Zhang1, Pei-Pei Zhang1,Wen-Ping Zhou1, Jia-Yu Yu2, Zhi-Hua Yao1, Jun-Feng Chu1, Shu-Na Yao1, Cheng Wang2, Waseem Lone2, Qing-Xin Xia3, Jie Ma3, Shu-Jun Yang1, Kang-Dong Liu4,5, Zi-Gang Dong4, Yong-Jun Guo3, Lynette M. Smith6, Timothy W. McKeithan7, Wing C. Chan7, Javeed Iqbal2, and Yan-Yan Liu1

Abstract

Purpose: One third of patients with diffuse large B-cell an independent prognostic factor for RCHOP resistance after lymphoma (DLBCL) succumb to the disease partly due to adjusting for International Prognostic Index, cell-of-origin rituximab resistance. Rituximab-induced calcium flux is an classification, and MYC/BCL2 double expression. Loss of important inducer of apoptotic cell death, and we investigated CACNA1C expression reduced rituximab-induced apoptosis the potential role of calcium channels in rituximab resistance. and tumor shrinkage. We further demonstrated direct inter- Experimental Design: The distinctive expression of calcium action of CACNA1C with CD20 and its role in CD20 stabi- channel members was compared between patients sensitive lization. Functional modulators of L-type calcium channel and resistant to rituximab, cyclophosphamide, vincristine, showed expected alteration in rituximab-induced apoptosis doxorubicin, prednisone (RCHOP) regimen. The observation and tumor suppression. Furthermore, we demonstrated that was further validated through mechanistic in vitro and in vivo CACNA1C expression was directly regulated by miR-363 studies using cell lines and patient-derived xenograft mouse whose high expression is associated with worse prognosis in models. DLBCL. Results: A significant inverse correlation was observed Conclusions: We identified the role of CACNA1C in ritux- between CACNA1C expression and RCHOP resistance in two imab resistance, and modulating its expression or activity may independent DLBCL cohorts, and CACNA1C expression was alter rituximab sensitivity in DLBCL.

Introduction abnormalities, biological features, and prognosis (1, 2). The addition of rituximab, a humanized chimeric anti-CD20 mono- Diffuse large B-cell lymphoma (DLBCL) is the most common clonal antibody, to the standard CHOP chemotherapy (RCHOP) type of non-Hodgkin lymphoma characterized by distinct genetic has significantly improved overall survival (OS) of DLBCL patients (3). However, 30% to 40% of patients experience incom- 1Department of Internal Medicine, Affiliated Cancer Hospital of Zhengzhou plete response during treatment or progression after the comple- University and Henan Cancer Hospital, Zhengzhou, Henan, China. 2Department tion of RCHOP treatment (4). Salvage therapy and/or autologous of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, stem cell transplantation do not improve the dismal prognosis, Nebraska. 3Department of Molecule and Pathology, Affiliated Cancer Hospital of especially in patients with prior rituximab treatment and early Zhengzhou University and Henan Cancer Hospital, Zhengzhou, Henan, China. relapse (5). It is often regarded that rituximab resistance plays a 4China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China. 5Depart- vital role in treatment failure, thus requires further understand- ment of Pathophysiology, Basic Medical College, Zhengzhou University, fi Zhengzhou, Henan, China. 6Department of Biostatistics, University of Nebraska ings to improve clinical ef cacy of rituximab. Rituximab induces Medical Center, Omaha, Nebraska. 7Department of Pathology, City of Hope cell death via antibody-dependent cell-mediated cytotoxicity National Medical Center, Duarte, California. (ADCC), complement-dependent cytotoxicity (CDC), direct apo- Note: Supplementary data for this article are available at Clinical Cancer ptotic signaling, and possible vaccinal effects (6). It can inhibit the Research Online (http://clincancerres.aacrjournals.org/). expression of the antiapoptotic genes BCL2/BCL-XL by down- – J.-Y. Zhang, P.-P. Zhang, and W.-P. Zhou contributed equally to this article. regulating the survival pathways (refs. 7 9; i.e., p38MAPK, NF-kB, ERK 1/2, and PI3K-AKT). The resistance to rituximab is either Corresponding Authors: Yan-Yan Liu, Affiliated Cancer Hospital of Zhengzhou tumor-intrinsic (i.e., loss of CD20 expression; ref. 10) and/or University, 127 Dong Ming Road, Zhengzhou 450008, Henan, China. Phone: 1008613838176375; Fax: 1008637165588134; E-mail: [email protected]; host-associated factors (FcRIII-a polymorphism at amino acid Javeed Iqbal, Department of Pathology and Microbiology, LTC-11714, Zip 7760, position 158) that inhibit ADCC action (11). Novel anti-CD20 University of Nebraska Medical Center, Omaha, NE 68105. E-mail: [email protected]; antibodies (e.g., Obinutuzumab) have been developed to exceed and Wing C. Chan, City of Hope National Medical Center, Department of Pathology, rituximab action (12, 13), but with limited improvement for Familian Science Building, Room 1215, 1500 East Duarte Rd, Duarte, CA 91010. DLBCL prognosis. E-mail: [email protected]. CD20 belongs to the MS4A gene family, having a tetraspanning doi: 10.1158/1078-0432.CCR-18-2146 membrane structure with an N- and C-terminal cytoplasmic 2019 American Association for Cancer Research. domain involved in B-cell proliferation (14). CD20 has been

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Table 1. Comparison of clinical characteristics between patients sensitive and Translational Relevance resistant to RCHOP regimen B cells express a wide and diverse pool of ion channels, Resistance Sensitivity Characteristics (n ¼ 43) (n ¼ 68) P value including components of the Cav1 subfamily of voltage-gated Gender L-type calcium channels. L-type calcium channels can be Male, n (%) 20 (47) 32 (47) 0.955 manipulated by many small molecules and are capable of Age, median (range, y) 41 (19–82) 47 (18–80) 0.195 mediating calcium influx, which is required to trigger apopto- Performance status sis. Crosslinking of CD20 molecules by rituximab can induce ECOG score 0–1, n (%) 33 (77) 60 (88) 0.092 calcium influx leading to apoptosis. In this study, we identified Ann Arbor stage n the role of L-type Cav 1.2 Calcium Channel-a-1C (CACNA1C) I/II, (%) 20 (47) 42 (62) 0.115 LDH level in regulating response to rituximab in diffuse large B-cell Normal, n (%) 23 (53) 44 (65) 0.239 lymphoma. These observations provide a rationale that mod- Number of extranodal involvement ulating L-type Cav1.2 calcium channel may increase the clin- More than 1, n (%) 5 (12) 6 (9) 0.630 ical efficacy of rituximab. Actually, more attention has now IPI score been paid to calcium channels due to their involvement in the Low/low-intermediate risk 0–2, n (%) 30 (70) 60 (88) 0.016 proliferation and invasion of cancer cells and their potential Cell origin Germinal-center B cell, n (%) 11 (26) 20 (29) 0.661 modulation by pharmaceuticals. CACNA1C expression, n (%) 19 (44) 52 (76) 0.001 Double expression, n (%) 9 (21) 5 (7) 0.044

considered as a component of a cell-surface calcium channel or as a direct regulator of calcium channel activity (15, 16). Calcium Materials and Methods channels/pumps located in the plasma membrane and subcellu- Patients and cell lines lar organelles can modulate intracellular calcium signaling. Cross- The study was carried out in a retrospective series of 48 DLBCL linking of CD20 molecules by rituximab induces a redistribution cases with cryopreserved tissues and 63 cases with formalin-fixed of CD20 molecules to lipid rafts at the plasma membrane and paraffin-embedded tissues. Basic clinical characteristics of calcium influx leading to apoptosis (17). The increased intracel- patients are presented in Table 1. The diagnosis of DLBCL was lular calcium induced by rituximab was demonstrated to depend confirmed by at least two pathologists in accordance to the World on the calcium channel at the plasma membrane, rather than Health Organization classification (25). All patients were treated those at the endoplasmic reticulum (18). CD20 deficiency sig- with the RCHOP regimen, and involved-field radiotherapy was nificantly reduced ligation-induced intracellular calcium performed as consolidation treatment in 5 cases in the primary responses (19). However, CD20-deficient B cells presented therapy. Responses to treatment were evaluated by CT scans or incomplete blockade of calcium responses (14), which reflect the PET/CT following the response criteria for lymphoma as defined possibility that other calcium channels may participate in regu- by Cheson and colleagues (26). The study was reviewed and lating calcium influx. approved by hospital review boards with informed consent of B lymphocytes express a wide and diverse pool of ion channels, the patients and was conducted in accordance with the Declara- including components of the Cav1 subfamily of voltage-gated tion of Helsinki. Another independent DLBCL cohort (27) was L-type calcium channels (VGCC) which are signature molecules of used to validate the findings. excitable cells (20, 21). L-type calcium channels are composed of OCI-ly7, OCI-ly8, and OCI-ly3 DLBCL cell lines were cultured five subunits, including a1, a2/d, b, and g. There are four different in IMDM (Gibco) with 10% FBS, 100 U/mL penicillin, 100 mg/ pore-forming a1 subunits, Cav1.1 to 1.4, being encoded by mL streptomycin, and 0.2% beta mercaptoethanol (Invitrogen). calcium channel alpha 1S (CACNA1S), CACNA1C, CACNA1D, These cell lines were gifted from Department of Pathology and and CACNA1F genes, which mediate L-type currents and contain Microbiology, University of Nebraska Medical Center. All of them drug-binding sites. Its other subunits primarily modify channel were genotyped by short tandem repeat analysis and were con- gating or affinity, being encoded by CACNA2D1-4, CACNB1-4, firmed to be negative for mycoplasma at the time of testing with and CACNG1-8. These channels in B lymphocytes were proven to the mycoplasma detection Kit (Lonza). play roles on calcium influx and cell proliferation by inhibitors of L-type channels (22). Indeed, L-type calcium channels can be fi manipulated by small molecules and are capable of mediating RNA isolation and gene expression pro ling fi long-lasting calcium influx, which are required in triggering Total RNA for gene expression pro ling (GEP) was extracted apoptosis (23). We hypothesized that enhancing calcium influx using the Qiagen all prep RNA and DNA isolation Kit (Qiagen). of B lymphoma cells may increase clinical efficacy of rituximab. In We used HG-U133 plus 2 arrays (Affymetrix) for GEP, and RNA the current study, we have tested the role of L-type calcium processing, hybridization, and image processing were performed channels in modulating rituximab-induced apoptosis and resis- according to the protocol described previously (28). The expres- miR-363 tance to initial therapy in DLBCL, and identified critical role of sion of for these cases has been quantitated in a previous CACNA1C expression in DLBCL prognosis. Furthermore, the study (24). biological role of CACNA1C was investigated using in vitro, in vivo lymphoma models, which demonstrated direct association qRT-PCR of CD20 with CACNA1C in plasma membrane and also regula- For qRT-PCR, total RNA was isolated from tissues and cells tion of CACNA1C expression by prognostically relevant miR-363 using the miRNeasy Mini Kit (Qiagen). Note that 2 mg RNA was in DLBCL (24). transcribed with the High Capacity cDNA Reverse Transcription

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Kits (ABI) according to the manufacturer's instructions. The qRT- cytometry. The Fluo-4 bound cytoplasmic free calcium and emit- PCR reactions were set up in triplicate using Brilliant II SYBR ted a green fluorescence (peak at 526 nm) detected using FL1. Green qPCR Master Mix (Toyobo) and ran on an ABI PRISM7300 Real-Time PCR system (Applied Biosciences) with the specific Western blotting assay primers (Supplementary Table S1). Total proteins were extracted with NP40 lysis buffer (Beyo- time). Western blotting assay was carried out using standard IHC assay protocols with antibodies against CACNA1C (Omnimabs, Indirect immunoperoxidase assays were performed on 5-mm- 1:500), CACNA1D (Omnimabs, 1:200), CACNA1F (Abcam, thick paraffin sections using antibodies against CACNA1C 1:1,000), CACNA1S (Abcam, 1:2,000), and CD20 (Abcam, (Omnimabs, 1:100), CD20 (Abcam, 1:50), BCL2 (Abcam, 1:2,000). Protein bands were visualized using the enhanced 1:250), or C-MYC (Abcam, 1:250). Antigen retrieval was per- chemiluminescence system (Millipore) according to the manu- formed by high pressure heating for 1 minute in PH 6.0 buffer for facturer's instruction. CACNA1C and in PH 9.0 buffer for CD20, BCL2, and C-MYC. Both CACNA1C and CD20 were located at plasma membrane, Coimmunoprecipitation and the positive one was defined to have more than 30% of Total proteins from OCI-ly7 cells were extracted with NP40 lymphoma cells stained. lysis buffer (Beyotime). Anti-CD20 antibody (Abcam, 1:50) or control immunoglobulin (anti-IgG; Santa Cruz Biotechnology, Flow cytometry assay 1:250) was incubated with cell lysate. Antigen–antibody complex The cells were fixed with 80% methanol for 5 minutes was slowly shaken on rotating shaker overnight at 4C followed and then permeabilized with PBST (0.2% Tween-20) for 20 min- by protein A/G PLUS-Agarose (Santa Cruz Biotechnology, 20 mL/ utes. The cells were incubated in 10% normal goat serum 500 mL) incubation for 3 hours at 4C. The pellets were washed 3 and followed by antibody against CACNA1C (Omnimabs, times with NP40 lysis buffer (Beyotime). The pulled-down pro- 1 mg/106 cells) for 30 minutes at 22C. The goat anti-mouse teins were examined by Western blotting as described above. IgG (HþL) antibody conjugated with FITC (ThermoFisher, 1:200) was added for 30 minutes at 22Cinthedark.The Construction of CACNA1C shRNA and miR-363 lentiviral fluorochrome-conjugated antibody against CD20 (BD, 1:20) vectors and transduction was incubated for 30 minutes at 22C in the dark. Run and The shRNA to suppress the expression of CACNA1C was analyze on flow cytometry. inserted into the Plk0.1-puro vector digested by AgeI/EcoRI according to the manufacturer's instruction. The lentiviral vector Apoptosis assay of miR-363 was generated after BamHI/EcoRI cloning and under After drug treatment, 1 106 cells were washed with PBS and the control of the Ubc promoter (Supplementary Fig. S1A). resuspended in the 500 mLof1 binding buffer containing 293T cells were transfected with 12 mg of plasmid containing Annexin-V–PE (Calbiochem). After incubation for 15 minutes at the desired construct and 8 mg of packing vector pSPAX2 and room temperature in the dark, cells were pelleted and resus- 4 mg of envelope vector pMD2G in a 10 cm dish for the pended in 500 mL binding buffer containing 10 mL 7-AAD, and production of lentivirus. After 48 hours, culture supernatant analyzed on a FACScan. The lower right-hand and the upper right- was filtered through the 0.45 mm filter and virus concentrated hand quadrant cells were considered apoptotic. with PEG 6000 method. Lymphoma cells were incubated with the virus preparation overnight, and the culture medium Immunofluorescence assay was replaced with 2 mL fresh medium. We used puromycin After being cultured with or without rituximab (50 mg/mL), (2 mg/mL) to select infected cells. OCI-ly7 cells were incubated with antibodies against CD20 (Abcam, 1:50) and CACNA1C (Omnimabs, 1:50) at 4C over- CRISPR/Cas9 system to knock out miR-363 night. Then the secondary antibodies Alexa Fluor 647 (Thermo The single-guide RNA (sgRNA) was designed nearest to the seed Fisher Scientific, 1:1,000) and Cy3 (Thermo Fisher Scientific, region of miR-363-3p and inserted into the pSpCas9 (BB)-2A-GFP 1:1,000) were incubated in the dark for 1 hour at room temper- (PX458) vector. A single-stranded DNA oligonucleotide (ssODN) ature, following 1 mg/mL DAPI being incubated in the dark for 1 of 99 bp was also designed in which the seed region of miR-363 minute. Finally, samples were added a drop of Dako fluorescence (ATTGCAC) was replaced by EcoRI sequence (GAATTC). The mounting medium (Dako) and mounted with coverslips. Images sgRNA and ssODN were cotransfected into OCI-ly3 cell line using were collected with Nikon confocal microscope system. the Lonza Nucleofector system (Supplementary Fig. S1B). EGFP- positive cells were sorted by flow cytometry after transfection for Calcium flux assay 48 hours and then diluted into 96-well to generate single clones. OCI-ly3 and OCI-ly7 cells were suspended at a density of 1 All single clones were examined by DNA sequencing and qRT-PCR 106 cells/mL with the Hank's Balanced Salt Solution (HBSS) before further application. containing 1% BSA(W/V), and 2 mmol/L Fluo-4 AM was dis- patched into the 5 mL Falcon cytometry tubes for 30 minutes at Xenograft mouse model assay 37C in the dark. After being washed twice with HBSS, the cells Five- to 7-week-old SCID mice (Charles River) were injected were resuspended in saline solution containing 10 mmol/L with 1 107 OCI-ly7 cells that had been transduced with e-GFP or HEPES, 1 mmol/L Na2HPO4, 137 mmol/L NaCl, 5 mmol/L KCl, CACNA1C shRNA lentiviral vector, or DLBCL cells from patient 1 mmol/L CaCl2, 0.5 mmol/L MgCl2, 5 mmol/L Glucose, and sample in the posterior flank subcutaneously. When the tumor 0.1% BSA(W/V). After adding 50 mg/mL rituximab, calcium flux approached about 100 mm3, mice were randomly divided in assays were performed on the Becton-Dickinson FACS AriaII flow groups of seven mice each. Saline (control), rituximab (25 mg/g),

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nimodipine (2 mg/g), Bay K8644 (2 mg/g), rituximab (25 mg/g) divided into quartiles, the first quartile (Q1) with the lowest plus nimodipine (2 mg/g), and rituximab (25 mg/g) plus Bay expression showed independent association with RCHOP resis- K8644 (2 mg/g) were injected i.p. into patient-derived xenograft tance (OR ¼ 2.897, P ¼ 0.014). We also demonstrated the (PDX) mice once every other day for 10 times, respectively; saline independent correlation of the lowest quartile of CACNA1C (control) and rituximab (25 mg/g) were injected i.p. into OCI-ly7– expression with inferiors OS (HR ¼ 1.974, P ¼ 0.020; Fig. 1E). derived mouse xenografts once every other day for 7 times. Tumor Similar trends were observed with EFS analysis (HR ¼ 1.686, P ¼ volume was measured every 3 days with a caliper and calculated 0.053). In patients treated by CHOP regimen, we did not observe using the formula: (Length Width2) 0.5. Animals were the relation of CACNA1C expression with outcome (Supplemen- maintained in accordance with the principles of laboratory ani- tary Fig. S3). mal care under an Institutional Animal Care and Use Committee– approved protocol. Loss of CACNA1C expression leading to rituximab resistance To demonstrate the significance of CACNA1C expression in Statistical analysis rituximab resistance, we used three DLBCL cell lines OCI-ly3, Statistical analyses were performed using the Statistical OCI-ly7, and OCI-ly8 for in vitro assays, including qRT-PCR, flow Software Package for the Social Sciences (SPSS version 13.0 cytometry, and Western blotting assays. A good concordance was for Windows) and TIGR TM4 software (version 4.8.1). OS observed between mRNA and protein expression for CACNA1C and event-free survival (EFS) were estimated using the and CD20 with OCI-ly3 showing the lowest and OCI-ly7 showing Kaplan–Meier method, and differences were assessed using the highest expression (Supplementary Fig. S4A–S4E). When the log-rank test. We used the probe sets (242973_at and these cell lines were treated with rituximab (50 mg/mL) for 48 238636_at) for estimating the CACNA1C mRNA expression hours, we observed a positive relationship between CACNA1C and rituximab efficacy association (Supplementary Fig. S2). expression and rituximab sensitivity. As shown in Supplementary Multivariate logistic regression examined the effect of CAC- Fig. S4F–S4G, cell lines with high expression of CACNA1C pro- NA1C mRNA expression on RCHOP resistance. Differences duced significant apoptosis (27.59% 2.41% in OCI-ly7, among groups were considered statistically significant at 18.84% 2.63% in OCI-ly8) compared with low expressing cell P values below 0.05 (). line (1.54% 1.39% in OCI-ly3; P < 0.05). To further validate mechanistically, we generated CACNA1C- deficient stable cell lines using shRNAs (sh1C-1 and sh1C-2) in Results OCI-ly7 and OCI-ly8 cell lines, which resulted in significant loss Inverse correlation between CACNA1C expression and RCHOP of CACNA1C protein expression, and observed significant resistance decrease in percentage of apoptosis in transduced cells after ritux- DLBCL patients treated with RCHOP regimen were divided imab treatment (OCI-ly7: 23.05% 2.12%, sh1C-1 9.85% into sensitive (n ¼ 30) and resistant (n ¼ 18) groups according 1.32%, P ¼ 0.042; sh1C-2 3.70% 1.03%, P ¼ 0.022; and to treatment response. Resistant patients were defined as not OCI-ly8: 17.86% 2.32%, sh1C-1 4.80% 1.06%, P ¼ 0.035; achieving complete remission or developing rapid disease sh1C-2 4.84% 1.08%, P ¼ 0.038; Fig. 2A and B). To validate progression (less than 6 months) after 6 to 8 cycles of RCHOP the finding in vivo, xenografts mice models were established administration. Using GEP, we found that members of L-type using transduced OCI-ly7 cell line (with and without sh1C-1 calcium channel [i.e., CACNA1 (-C, -D, -F, -S), CACNA2 (-D1, vector) and treated with rituximab. As expected, xenografts with -D2, -D3,-D4), CACNB (-1,-2,-3,-4), CACNG (1to8)] were control vector showed significant tumor shrinkage in tumor vol- expressed in both sensitive and refractory subgroups. Of them ume (P ¼ 0.035) and weight (P ¼ 0.024), whereas those with only CACNA1C mRNA expression presented significant differ- sh1C vector was not significantly reduced in tumor volume (P ¼ ence with lower levels in resistant patients (P ¼ 0.009), whereas 0.273) and weight (P ¼ 0.278; Fig. 2C). These data suggested CD20 mRNA expression did not show significant difference that loss of CACNA1C significantly reduced rituximab-induced (Fig. 1A; Supplementary Table S2). The CACNA1C expression tumor inhibition. was not significantly different between germinal center (GC) B-cell (n ¼ 26) and non-GC B-cell (n ¼ 22) subtypes (P ¼ Direct interaction of CACNA1C with CD20 during rituximab 0.797). Using IHC, we observed that loss of CACNA1C protein action was significantly associated with RCHOP resistance (44% vs. Using confocal microscopy, we observed that CD20 molecules 76%, P ¼ 0.001; Table 1). Two cases which represented the located in the plasma membrane exhibited a gradual polarization distinctive expression were presented in Fig. 1B. Univariate (green light) after rituximab treatment at 0, 2, and 12 hours analysis revealed the correlation of CACNA1C expression (Fig. 3A). CACNA1C molecule mainly confined to the plasma (P ¼ 0.001), double expression (P ¼ 0.044), and international membrane with modest cytoplasmic and nuclear expression, and prognostic index (IPI) score (P ¼ 0.017) with resistant disease. showed similar polarized distribution (red light) after rituximab Multivariate analysis showed the independent association of treatment. The colocalization of CD20 and CACNA1C was CACNA1C expression and IPI score with resistance, excluding observed in the merged image with overlapping distribution double expression. (yellow light) in the plasma membrane, which suggested possible The results were confirmed in an independent DLBCL cohort interaction. To validate their interaction, CACNA1C protein was with RCHOP treatment (27). There was no correlation between coimmunoprecipitated with either anti-CD20 or anti-IgG anti- CACNA1C expression and cell-of-origin classification (P ¼ body after rituximab treatment of 2 hours in OCI-ly7 cells. 0.94; Fig. 1C). Of the normal B-cell subsets, centrocytes and CACNA1C protein was detected in anti-CD20 pull-down pro- plasmablast cells showed comparatively lower expression of teins, suggesting the association between CD20 and CACNA1C CACNA1C mRNA (Fig. 1D). When CACNA1C expression was molecules (Fig. 3B).

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Figure 1. CACNA1C expression and signiﬁcance in DLBCL patients and normal B cells. A, The mRNA expression of CACNA1C was lower in rituximab-resistant patients compared with rituximab-sensitive patients (P ¼ 0.009), whereas CD20 mRNA expression showed no correlation (P ¼ NS). B, The protein expression of CACNA1C was negative in one case with rituximab resistance and positive in the other case with rituximab sensitivity, whereas CD20 protein expression was positive in both of them. C, In an independent DLBCL cohort, there was no signiﬁcant difference in CACNA1C mRNA expression between GCB-DLBCL and ABC- DLCBL subtypes (P ¼ NS). D, Of the normal B-cell subsets, centrocytes and plasmablast showed comparatively lower expression of CACNA1C. E, The patients with lowest quartile of CACNA1C expression showed inferior OS (HR ¼ 1.974, P ¼ 0.020).

Because rituximab performs its action via the ligation with Modulators of L-type calcium channel affecting rituximab cell surface CD20 molecule, we further explored the role of efficacy CACNA1C in regulating rituximab action. Knockdown of CAC- Because there was not a specific modulator of CACNA1C, NA1C expression in DLBCL cell lines downregulated CD20 antagonist (nimodipine) and agonist (Bay K8644) of pan-L-type proteinexpressionwithoutanysignificant change in CD20 calcium channel were used to evaluate the effect of on rituximab mRNA levels (Fig. 3C). Other forms of a1 subunits, such as efficacy. We observed significant reduction of apoptosis in cell CACNA1D, CACNA1F, and CACNA1S, showed no obvious lines when treated with rituximab (50 mg/mL) and nimodipine alteration, indicating the specificeffectofCACNA1ConCD20 (2 mmol/L) for 48 hours compared with rituximab alone (OCI-ly7: expression. When these cells were treated with the proteasome 26.02% 3.12% vs. 10.05% 1.25%, P < 0.01; OCI-ly8: 17.65% inhibitor bortezomib (20 nmol/L) for 6 hours, CD20 expres- 1.54% vs. 1.89% 0.98%, P < 0.01). When rituximab com- sionwasrestoredsuggestingthatCACNA1Cmayplayarolein bined with agonist Bay K8644 (10 nmol/L), significant increase in proteasome-dependent CD20 degradation, thus affecting effi- apoptosis was observed in the above cell lines (OCI-ly7: 26.02% cacy of rituximab (Fig. 3D). 3.12% vs. 37.36% 3.18%, P < 0.01; OCI-ly8: 17.65% 1.54%

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Figure 2. The effect of CACNA1C downregulation on rituximab- induced apoptosis and tumor inhibition. A and B, The interference with shRNA to CACNA1C reduced the percentage of rituximab- induced cell apoptosis (P < 0.05) in OCI-ly7 and OCI-Ly8 cell line. C, In xenograft mice models of OCI-ly7 cell line, downregulation of CACNA1C by its shRNA obviously suppressed rituximab-induced tumor shrinkage in tumor volume and weight (P < 0.05).

vs. 35.02% 2.58%, P < 0.01), and even in OCI-ly3 cell line, in significant increase in intracellular calcium ions in OCI-ly3 and which initially showed poor response to rituximab (1.54% OCI-ly7 cell lines. Expectedly, rituximab-mediated intracellular 1.39% vs. 21.13% 4.39%, P < 0.01; Fig. 4A). calcium ions were enhanced in cells treated with Bay K8644 for 2 Because rituximab-mediated calcium influx is a sensitive indi- hours, whereas it was reduced in cells treated with nimodipine for cator of rituximab action, we measured the calcium influx with 2 hours. Ionomycin (2 mg/mL) was used as a positive control for fluo-4-AM using flow cytometry after rituximab (50 mg/mL) calcium influx (Supplementary Fig. S5A). Neither nimodipine nor treatment. We observed that the addition of rituximab resulted Bay K8644 treatment alone led to a significant alteration in

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Figure 3. The interaction between CACNA1C and CD20 molecules during rituximab action. A, CD20 (green light) and CACNA1C (red light) molecules exhibited a similar polarized distribution after rituximab treatment of 0, 2, and 12 hours. Their overlapping distribution (yellow light) was captured in the plasma membrane in the merged phase. B, CACNA1C protein was detected in anti-CD20 pull-down protein using Western blot after rituximab treatment of 2 hours in OCI-ly7 cells. C, Knockdown of CACNA1C expression with transduced shRNAs did not affect CD20 mRNA levels, but downregulated CD20 protein expression. Other isoforms of a1 subunits, such as CACNA1D, CACNA1F, and CACNA1S, showed no obvious alteration. D, Proteasome inhibitor bortezomib (20 nmol/L) restored CD20 expression after administration of 6 hours in OCI-ly7 cells.

intracellular calcium ions. Concurrently, we did not ﬁnd the nor modulators of L-type calcium channel alone showed obvious inﬂuence of nimodipine and Bay K8644 on CD20 expression effect on tumor growth. after treatment of 24 and 48 hours in the study (Supplementary Fig. S5B). MiR-363 epigenetically regulating the expression of We tested the action of these compounds in in vivo DLBCL PDX CACNA1C mice (Fig. 4B) and observed that rituximab combined with Target Scan, a computational algorithm, predicted CACNA1C agonist (Bay K8644) markedly enhanced rituximab action, as to be the direct target of miR-363. We demonstrated the direct estimated by tumor volume (P ¼ 0.024) and weight (P ¼ 0.012). regulation of miR-363 on CACNA1C expression using luciferase Neither rituximab combination with antagonist (nimodipine) reporter assay in 293T cells, where we found that luciferase

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Figure 4. The inﬂuence of L-type calcium channel modulators on rituximab- induced apoptosis and tumor suppression. A, The agonist of L-type calcium (Bay K8644) enhanced the rituximab-induced apoptosis, whereas the antagonist of L-type calcium channel (nimodipine) attenuated the rituximab-induced apoptosis (P < 0.01) in DLBCL cell lines. B, In PDX mouse models, rituximab combined with agonist (Bay K8644) markedly enhanced rituximab action (P < 0.05). Neither its combination with antagonist (nimodipine) nor modulators of L-type calcium channel alone showed obvious effect on tumor growth (P > 0.05).

expression was significantly inhibited by miR-363 with wild-type 30-untranslated region (UTR) of CACNA1C, but not with mutant Discussion 30-UTR of CACNA1C (Fig. 5A). Furthermore, ectopic expression of We investigated the role of L-type calcium channel in ritux- miR-363 in OCI-ly7 and OCI-ly8 cell lines led to significant loss of imab-induced apoptosis and demonstrated that loss of CAC- CACNA1C and as anticipated loss of CD20 protein expression in NA1C expression correlates with rituximab-mediated immuno- these cell lines (Fig. 5B). The overexpression of miR-363 signifi- chemotherapy resistance in DLBCL. The role of CACNA1C in cantly reduced rituximab-induced apoptosis in OCI-ly7 (27.07% normal B-cell activation is not known, but several studies showed 2.54% vs. 1.22% 1.06%, P ¼ 0.018) and OCI-ly8 cell lines that L-type calcium channels play a role in B-cell calcium influx (17.23% 2.21% vs. 2.94% 1.11%, P ¼ 0.014) after rituximab and proliferation (22), and increased intracellular calcium corre- treatment of 48 hours (Fig. 5C). To explore direct regulation, we lates with apoptosis (29). Calcium influx can affect the depolar- furtherknockedoutmiR-363locusintheOCI-ly3celllineusingthe ization of mitochondria, which is fundamental in the activation CRISPR/Cas9 system, and as expected resulted in the upregulation of executioner caspases (30). The increased levels of cytosolic of CACNA1C (Fig. 5D). These edited cells showed significantly calcium may also activate calcium-dependent proteases, such as increased apoptosis (0.80% 0.69% vs. 10.25% 1.80%, P ¼ calpains, which have been implicated in caspase-independent 0.001; Fig. 5E), when treated with rituximab. death (31). The elevated intracellular calcium has been shown

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L-Type Calcium Channel in Diffuse Large B-Cell Lymphoma

Figure 5. The association of miR-363 with CACNA1C expression and rituximab-induced apoptosis. A, The miR-363 inhibited the luciferase activity in 293T cells with wild-type (WT) 30-UTR of CACNA1C gene, but did not affect it in cells with mutant 30-UTR of CACNA1C gene (P < 0.01). B, The ectopic expression of miR-363 reduced CACNA1C and CD20 protein expression in OCI-ly7 and OCI-ly8 cell lines. C, The ectopic expression of miR-363 decreased rituximab-induced apoptosis in OCI-ly7 and OCI-ly8 cell lines (P < 0.05). D, The knockout of miR-363 led to the upregulation of CACNA1C expression and (E)the increase of rituximab-induced apoptosis (P < 0.05).

to be critical in rituximab-induced apoptosis. Thus, enhancing death. This direct relationship between CACNA1C expression and calcium influx could be an approach to increase rituximab- rituximab sensitivity was also demonstrated in DLBCL cell lines induced direct cell apoptosis in DLBCL. We attempted to knock and xenograft mouse models. out CACNA1C using CRISPR/Cas9 system in DLBCL cell lines, but Rituximab resistance remains to be a clinical challenge. Many these knockout cells lost the ability of persistent growth (Supple- studies have explored the regulatory mechanisms of CD20 expres- mentary Fig. S6), which indicated a critical role of CACNA1C in sion, including transcription factors SPI1/PU.1 (32), Oct2 (33), the long-term survival of DLBCL cells. and Pax5 (34). Other factors, such as epigenetic changes, somatic The independent association of CACNA1C expression with mutation, and noncoding RNA, could also affect CD20 expres- refractory disease and inferior survival was proven in DLBCL sion (35). A recent study using RNAi screening method identified patients. We did not find distinctive expression of CACNA1C CREM (cAMP Responsive Element Modulator) as a top candidate between GCB and ABC DLBCL subtypes. Of the normal B-cell for CD20 suppression, CHD4 (Chromodomain Helicase DNA subsets, centrocytes and plasmablast cells showed comparatively Binding Protein 4) and MBD2 (Methyl-CpG Binding Domain lower CACNA1C expression. Our study further revealed the Protein 2) as positive regulators of CD20 expression (36). In function of the accessory L-type calcium channels CACNA1C in addition, CD20 regulation at the posttranscriptional level was rituximab-induced apoptosis, and the interaction between CAC- also suggested. Bortezomib can sensitize CD20-positive lympho- NA1C and CD20 in cell membrane is critical for this process. We ma cells to rituximab-induced CDC after the incubation of 24 observed direct interaction between CACNA1C and CD20 mol- hours (37), and rituximab-resistant lymphoma cells were ecule using confocal immunofluorescent and pull-down assays observed to exhibit upregulation of components of the ubiqui- and demonstrated that loss of CACNA1C expression resulted in tin-proteasome system (38, 39). In this study, we found that lower CD20 stability and also reduced rituximab-induced cell CACNA1C may perform a critical function in CD20 protein

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Zhang et al.

stabilization through proteasome system, because lowered CD20 tion of CACNA1C appeared to be also associated with reduced protein level was restored in cells with CACNA1C shRNAs after CD20 expression that may reduce target binding by rituximab. bortezomib incubation of 6 hours. Further studies on the interaction between CACNA1C and CD20 Furthermore, we investigated the effect of modulators of L-type in calcium flux in lymphoma may lead to discovery of novel calcium channel on rituximab-induced apoptosis and explored mechanisms of calcium ions transport in DLBCL that may have the potential approach to raise therapeutic efficacy for DLBCL in therapeutic implications. The effect of miR-363 on CACNA1C and the future clinical trials. This study validated that inhibiting L-type rituximab efficacy further extended our insight into the mechan- calcium channel with nimodipine reduced rituximab action, isms of refractory disease, and it may serve as a prognostic marker whereas Bay K8644, an agonist of L-type calcium channel, or novel therapeutic target in the future. increased it. These observations provide a rationale that modulating L-type Cav1.2 calcium channel may increase the clinical Disclosure of Potential Conflicts of Interest efficacy of rituximab. Actually, more attention has now been paid No potential conflicts of interest were disclosed. on calcium ion channels due to their involvement in the proliferation and invasion of cancer cells and their potential modula- Authors' Contributions tion by pharmaceuticals (40, 41). This is the first study showing Conception and design: J.-Y. Zhang, P.-P. Zhang, W.-P. Zhou, Z.-G. Dong, that modulating Cav1.2 subfamily of L-type calcium channel can J. Iqbal, Y.-Y. Liu Development of methodology: J.-Y. Zhang, P.-P. Zhang, W.-P. Zhou, J. Ma, affect rituximab-induced cell apoptosis on DLBCL. Blocking J. Iqbal, Y.-Y. Liu L-type calcium channels in human B cells has been demonstrated Acquisition of data (provided animals, acquired and managed patients, to reduce B-cell receptor (BCR)-induced calcium entry (42). Some provided facilities, etc.): J.-Y. Zhang, P.-P. Zhang, W.-P. Zhou, J.-Y. Yu, knowledge gaps on L-type calcium channels remain to be J.-F. Chu, S.-N. Yao, J. Ma, S.-J. Yang, Y.-J. Guo, J. Iqbal, Y.-Y. Liu addressed in B lymphocytes and malignancy. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The miR-363 is a member of miR-106-363 cluster which is also computational analysis): J.-Y. Zhang, P.-P. Zhang, W.-P. Zhou, J.-Y. Yu, Z.-H. Yao, C. Wang, W. Lone, Q.-X. Xia, S.-J. Yang, K.-D. Liu, L.M. Smith, a paralog of miR-17-92 superfamily (43–45). Although the – W.C. Chan, Y.-Y. Liu miRNA-17 92 cluster is a paradigm of oncomirs and cooperates Writing, review, and/or revision of the manuscript: J.-Y. Zhang, P.-P. Zhang, with c-Myc in the formation of mouse B-cell lymphoma (46), W.-P. Zhou, L.M. Smith, T.W. McKeithan, W.C. Chan, J. Iqbal, Y.-Y. Liu limited data are available on miR-106-363 cluster members. The Administrative, technical, or material support (i.e., reporting or organizing miR-106-363 cluster is mapped to chromosome X (47), and high data, constructing databases): J.-Y. Zhang, P.-P. Zhang, W.-P. Zhou, expression of the miR-106-363 cluster has been found in 46% of Z.-G. Dong, Y.-J. Guo Study supervision: J.-Y. Zhang, P.-P. Zhang, W.-P. Zhou, Z.-G. Dong, J. Iqbal human T-cell leukemias (48) and colon and prostate cancers (49) Other (pathology review; and scoring IHC stains): Q.-X. Xia suggesting its oncogenic potential. In our previous miRNAs profiling studies (24, 50), miR-363 was found to have a higher Acknowledgments expression in mantle cell lymphoma and was associated with Y.-Y. Liu is supported by the grants from the National Natural Science poor response to RCHOP regimen in DLBCL patients (24). How- Foundation of China (No. 81071938 and No. 81470365). W.C. Chan is ever, the mechanism by which miR-363 mediates the poor partially supported by NCI P30 CA033572 and NCI SPORE 2P50 CA107399. response to RCHOP remains elusive. This study demonstrated J. Iqbal is supported by the Lymphoma Research Foundation, the Leukemia and that miR-363 can directly downregulate the expression of CAC- Lymphoma Society, and the UNMC Clinical-Translational Research Scholars Program; partial support is from NCI Eppley Cancer Center Support Grant NA1C and may thus reduce rituximab-induced apoptosis. P30CA036727, National Center for Research Resources 5P20RR016469, and The relevance of miR-363 to RCHOP resistance will be further National Institute for General Medical Science 8P20GM103427. addressed in our future studies. In summary, the alteration of CACNA1C expression and mod- The costs of publication of this article were defrayed in part by the payment of ulators of L-type channel modulates rituximab-induced apopto- page charges. This article must therefore be hereby marked advertisement in sis. Notably, the agonist of L-type calcium channel, Bay K8644, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. has been shown to synergize in rituximab-induced apoptosis and tumor suppression in DLBCL. The optimal dose and safety of Bay Received July 6, 2018; revised November 28, 2018; accepted February 26, K8644 or its analogues are worth evaluating in the future. Reduc- 2019; published first March 1, 2019.

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L-Type Cav 1.2 Calcium Channel-α-1C Regulates Response to Rituximab in Diffuse Large B-Cell Lymphoma

Jiu-Yang Zhang, Pei-Pei Zhang, Wen-Ping Zhou, et al.

Clin Cancer Res Published OnlineFirst March 1, 2019.

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